The goal of this proposal is to investigate basic mechanisms regulating the uptake and efflux of lipoprotein cholesterol by macrophages. These studies are relevant to the process of macrophage foam cell formation and atherogenesis. Abnormalities in these processes that predispose to macrophage foam cell formation in diabetes will be explored. Recently, the ATP binding cassette transporter, ABCA1, was found to be responsible for Tangier Disease. ABCA1 promotes phospholipid and cholesterol efflux to apoA-I, and when mutated macrophages accumulate massive amounts of cholesterol. In Aims 1 and 2, we will evaluate novel mechanisms of post-transcriptional regulation of ABCA1 protein turnover and function. In Aim 1 we will determine the role of a recently identified PEST sequence within the ABCA1 molecule in regulating the rapid turnover and degradation of ABCA1, in an ubiquitin-regulated process. We will make mutants in the PEST sequence to determine if this affects ABCA1 protein levels and function. In Aim 2 the role of plasma membrane phospholipid fatty acid composition in regulating the ability of ABCA1 to mediate cholesterol efflux will be evaluated. We will determine the role of stearoyl CoA desaturase in regulating plasma membrane phospholipid fatty acid composition and test the hypothesis that increased activity of this enzyme decreases the formation of cholesterol-enriched plasma membrane microdomains that provide cholesterol for ABCA1-mediated efflux. These studies will involve the development of mice with specific macrophage over- or under-expression of SCD. In Aim 3 we will evaluate defects in macrophages of obese mice that cause decreased cholesterol efflux (increased SCD) and promote the uptake of oxidized LDL (increased CD36). Increases of SCD and CD36 have been observed in macrophages of obese mice, related to defects in macrophage insulin signaling. The role of increased SCD, increased CD36 and decreased macrophage insulin receptor activity in atherogenesis will be evaluated. This will include bone marrow transplantation experiments using macrophages from mice deficient in SCD, insulin receptors or CD36. The proposal will provide novel mechanistic information on macrophage foam cell formation, and will evaluate the hypothesis that defects in function of diabetic macrophages, related to altered insulin signaling, predispose to atherosclerosis.